13N isotope studies on the pathway of ammonia assimilation in Bacillus megaterium and Escherichia coli.
The pathway of ammonia incorporation into amino acids was studied by use of 13N-ammonium ions in Bacillus megaterium and Escherichia coli that had been grown aerobically on a minimal salts medium containing NH4Cl as the source of nitrogen. Anion- and cation-exchange high-pressure-liquid chromatography was used to separate amino acids relevant to the several possible pathways for ammonia assimilation in bacteria. At an initial concentration of added NH4+ of 1 microM, the glutamine synthetase-glutamate synthase pathway represented the major pathway in both bacteria on the basis of the effects of inhibitors of that pathway (L-methionine-DL-sulfoximine and azaserine) and of transamination (aminooxy-acetate) and the observation that the specific activity of glutamine was greater initially than that of any other amino acid likely to be the first product of an assimilation pathway. The study provides (i) a new analytical method for 13N-tracer investigation of amino acids, (ii) confirmation of conclusions from enzymological studies on the pathway of ammonia assimilation in B. megaterium and E. coli, and (iii) proof that alanine dehydrogenase and aspartate ammonia lyase (aspartase) are not important pathways in B. megaterium at low NH4+ concentrations.
D000596:Amino Acids / Q000096:biosynthesis; D000641:Ammonia / Q000378:metabolism*; D001410:Bacillus megaterium / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D009590:Nitrogen Radioisotopes; D011865:Radioisotope Dilution Technique; D013045:Species Specificity
D000596:Amino Acids; D009590:Nitrogen Radioisotopes; D000641:Ammonia